Use of bacteriophage outer membrane breaching proteins expressed in plants for the control of gram-negative bacteria

Abstract

The present invention provides compositions and methods for killing or suppressing growth of Gram-negative bacteria that infect, infest or cause disease in plants, including pathogenic, saprophytic and opportunistic microbes that cause disease in plants and food borne illness in people or in animal feed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims benefit as a Continuation-In-Part of U.S. application Ser. No. 10 / 556,563, filed Nov. 14, 2005, which claims benefit as a U.S. National Stage Application under 35 U.S.C. 371 of PCT / US2004 / 015099, filed May 14, 2004, which claims the benefit of U.S. Provisional Application Ser. No. 60 / 470,799, filed May 14, 2003, each of which are herein incorporated by reference in their entireties for all purposes. The present application also claims the benefit of U.S. Provisional Application No. 60 / 950,749, filed Jul. 19, 2007, which is herein incorporated by reference in its entirety for all purposes.DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY[0002]The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing of the Sequence Listing (filename: INTE 004 01US SeqList_ST25, date recorded: Jul. 21, 2008, file ...

AI Technical Summary

Benefits of technology

[0035]This invention provides: 1) methods for the identification of broad-spectrum BOMBs with a high level of nonenzymatic activity to breach microbial outer membranes and thereby increase the efficacy of both natural plant defense compounds and artificially applied compounds; 2) conditions required for maintaining and increasing the anti-microbial and anti-pest efficacy of BOMBs in gene fusions; 3) methods for effective targeting of BOMBs expressed in plants through use of a xylem enhanced promoter and a leader peptide to direct the BOMB protein to the plant apoplast and xylem; 4) methods for the control of Gram negative bacterial diseases of plants by expression of gene fusions involving BOMBs and BOMB fragments, C-terminal additions and leader peptides, and optionally, endolysins and / or lipases; 5) methods for increasing the shelf-life of cut flowers; and 6) transgenic plants useful for the production of novel antimicrobial proteins based upon BOMBs and BOMB fragments.

[0036]It has now been found by the present inventors that certain bacteriophage carry genes that encode proteins other than holins and endolysins that assist the phage in disrupting host cells, and specifically in disrupting the bacterial outer membrane or LPS layer found only in Gram negative bacteria. It has further been found that at least one such bacterial outer membrane breaching (BOMB) protein works from the outside of the cell to compromise the integrity of the bacterial LPS outer membrane. It has further been found that expression of a BOMB protein in Gram-negative bacteria inhibits the growth of the bacteria in culture, and that when coupled with detergents, lytic proteins such as certain lysozymes or plant defense compounds such as berberine chloride, growth inhibition and / or lysis occurs. Thus it has been discovered that a BOMB protein not only can have a direct inhibitory effect on Gram-negative bacteria in culture medium, but the effect is synergistic with enzymes that cause lysis or with compounds that are toxic. It has further been found that BOMB proteins compromise the integrity of the bacterial LPS barrier, but not the inner membrane. Further, the present inventors have: 1) identified, cloned and expressed Xanthomonas pelargonii phage Xp15 BOMB protein BC in E. coli; 2) operably fused the bombBC gene separately to plant promoters in a gene expression cassette; 3) expressed functional BombBC in multiple different transgenic plants, both monocot and dicot, including tomato, tobacco, geranium, citrus and rice; 4) killed or inhibited growth of many different Gram-negative pathogens of said plants, conferring enhanced disease resistance or immunity to said plants. Thus it has been discovered that BombBC, and more generally, BOMBs, may be functionally expressed in both monocot and dicot plants to enhance a plant's natural disease resistance mechanisms.

[0044]The present invention therefore also relates to a method for preparing transformed plant cells and plants, including seeds and all parts of plants, having increased resistance or immunity to Gram-negative bacterial infection or infestation, whether plant pathogenic or not. This method provides one or more BOMB genes, BOMB gene fusions, and the introduction of these genes and fusions into the genome of plant cells, followed by introduction of said genes into plant cells, regeneration of whole transformed plants from said cells, providing transgenic plants with resistance or immunity to disease, infection or infestation by Gram-negative bacteria. This invention describes the use of BOMB genes to control disease, infection and infestation in transgenic plants to: 1) control diseases otherwise affecting said transgenic plants, 2) to eliminate said transgenic plants from being carriers of diseases that affect other plants or animals (eg., nosocomial infestations or in animal feed), and 3) to prolong the shelf life of said transgenic plants if said plants are detached from roots (eg., cut flowers, grafting).

Problems solved by technology

Activity of BOMBs in destabilization of the outer membrane presumably allows natural defense molecules secreted by plants and / or by other microbes to also breach the outer membrane of the target cells, thereby compromising the “barrier function” of the Gram negative outer membrane.

Thus it has been discovered that a BOMB protein not only can have a direct inhibitory effect on Gram-negative bacteria in culture medium, but the effect is synergistic with enzymes that cause lysis or with compounds that are toxic.

It has further been found that BOMB proteins compromise the integrity of the bacterial LPS barrier, but not the inner membrane.

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